A Phytophthora receptor-like kinase regulates oospore development and can activate pattern-triggered plant immunity

Plant cell-surface leucine-rich repeat receptor-like kinases (LRR-RLKs) and receptor-like proteins (LRR-RLPs) form dynamic complexes to receive a variety of extracellular signals. LRR-RLKs are also widespread in oomycete pathogens, whereas it remains enigmatic whether plant and oomycete LRR-RLKs could mediate cell-to-cell communications between pathogen and host. Here, we report that an LRR-RLK from the soybean root and stem rot pathogen Phytophthora sojae, PsRLK6, can activate typical pattern-triggered immunity in host soybean and nonhost tomato and Nicotiana benthamiana plants. PsRLK6 homologs are conserved in oomycetes and also exhibit immunity-inducing activity. A small region (LRR5-6) in the extracellular domain of PsRLK6 is sufficient to activate BAK1- and SOBIR1-dependent immune responses, suggesting that PsRLK6 is likely recognized by a plant LRR-RLP. Moreover, PsRLK6 is shown to be up-regulated during oospore maturation and essential for the oospore development of P. sojae. Our data provide a novel type of microbe-associated molecular pattern that functions in the sexual reproduction of oomycete, and a scenario in which a pathogen LRR-RLK could be sensed by a plant LRR-RLP to mount plant immunity.

RLKs form the largest family of plant cell-surface receptors. Interestingly, LRR-RLKs with a similar domain structure are also widespread in oomycetes although they are evolutionarily distant to plants 15,16 . We previously reported that PcLRR-RK1 regulates the growth, development, and virulence of P. capsici 17 and several P. sojae LRR-RLKs play pivotal roles in zoospore production and virulence 16 .
LRR-containing proteins may interact with each other to regulate different biological processes. For MAMPs (microbe-associated molecular patterns) recognition, interactions between ECDs of LRR-RLKs are a prerequisite for ligand signal activation 18 . In Arabidopsis, an extracellular network consisting of 567 interactions among ECDs of 225 LRR-RLKs was identified 19 . A similar extracellular interactome of LRR proteins was revealed in Drosophila melanogaster 20 . Similar to plants and metazoans, P. sojae also has a complicated interaction network of LRR-RLKs 16 . Importantly, it is worth to notice that receptors with LRR ECDs between adjacent cells can also establish contacts and mediate cell-to-cell communication in metazoans 21 .
Given that both P. sojae and the host plant possess many LRR-RLKs that are essential for virulence or resistance, we hypothesized that they could mediate cell-to-cell communications during interaction. To test it, we systematically expressed 24 P. sojae LRR-RLKs and found that PsRLK6 could elicit immune responses in Nicotiana benthamiana. PsRLK6 is conserved and widespread across Peronosporales, whose homologs exhibited immune-inducing activity in the plant as well. Moreover, the immunity activated by PsRLK6 required two coreceptors BAK1 and SOBIR1, suggesting that an LRR-RLP binds PsRLK6 to mount plant immunity. Our data provide a novel type of microbeassociated molecular patterns (MAMP) that is a cell surface-localized RLK and a scenario showing plants and pathogens communicate with each other via cell-surface receptors.

P. sojae PsRLK6 can activate typical pattern-triggered immunity (PTI) in Nicotiana benthamiana
In our previous study, a total of 24 LRR-RLK genes (PsRLKs) were identified in the P. sojae genome 16 . We hypothesized that some PsRLKs could interact with plant receptors to induce plant immunity during infection. Thus, extracellular domains (ECDs) of all 24 PsRLKs were cloned and then transiently expressed in N. benthamiana by agroinfiltration. The DAB (3,3-diaminobenzidine) staining assay revealed that PsRLK6 ECD and PsRLK7 ECD could induce reactive oxygen species (ROS) accumulation in N. benthamiana, while other PsRLKs and the negative control GFP could not (Fig. 1a). To further examine the effect of PsRLK6 ECD and PsRLK7 ECD on plant resistance to pathogen, N. benthamiana leaves that expressed PsRLK6 ECD , PsRLK7 ECD , or GFP for 24 h were inoculated with P. capsici. Leaf regions expressing PsRLK6 ECD or PsRLK7 ECD showed significantly smaller lesion areas and less P. capsici biomass, compared to that expressing GFP ( Supplementary Fig. 1a, b). Western blot analysis showed that GFP, PsRLK6 ECD , and PsRLK7 ECD were successfully expressed at the expected size in N. benthamiana (Supplementary Figs. 1c, d). These results suggest that P. sojae PsRLKs could enhance plant resistance in N. benthamiana.
To further confirm that PsRLK6 ECD and PsRLK7 ECD could induce immune responses in N. benthamiana, we initially expressed and purified His-tagged PsRLK6 ECD and PsRLK7 ECD from Pichia pastoris ( Supplementary Fig. 2). The recombinant proteins were used to test for a series of PTI (pattern-triggered immunity) responses, including ROS burst, MAPK activation, and up-regulation of two PTI marker genes (PR1a and CYP71D20). As shown in Fig. 1b, 1 μM PsRLK6 ECD protein treatment increased ROS levels after about 5 min upon elicitation, reaching the maximum levels at 30 min. However, PsRLK7 ECD protein failed to induce detectable ROS production even at 60 min, similar to GFP protein (Fig. 1b). Therefore, PsRLK6 ECD was selected for further studies. To test whether PsRLK6 ECD -induced ROS burst is concentration-dependent, different concentrations of PsRLK6 ECD proteins ranging from 0.01 μM to 5 μM were examined. The result showed that the total ROS production of 60 min increased with higher concentrations (Fig. 1c).
MAPK activation is another early PTI (pattern-triggered immunity) signaling event. As shown in Fig. 1d, PsRLK6 ECD protein treatment triggered MAPK activation at 5, 10, and 15 min in N. benthamiana as detected by immunoblot with the α-pTEpY antibody. We also observed that the expressions of PTI marker gene PR1a and CYP71D20 were greatly activated by PsRLK6 ECD protein for 6 h (Fig. 1e, f). Finally, to examine the effect of PsRLK6 ECD protein on plant resistance to pathogen, N. benthamiana leaves were pre-treated with 1 μM PsRLK6 ECD or GFP proteins, respectively, and were inoculated with P. capsici 24 h later. Consistent with the above results, pre-treatment with PsRLK6 ECD protein resulted in significantly smaller lesions and less P. capsici biomass compared to GFP (Fig. 1g-i). Collectively, these findings show that PsRLK6 ECD can activate PTI (pattern-triggered immunity) responses in N. benthamiana.
BAK1 and SOBIR1 are required for PsRLK6-induced immune responses in N. benthamiana LRR-RLKs BAK1 and SOBIR1 are two co-receptors of most LRR-RLK and LRR-RLP receptors for signaling activation 4 . To determine whether PsRLK6 ECD -induced immune responses are BAK1-and SOBIR1dependent in N. benthamiana, we used the virus-induced gene silencing (VIGS) construct to silence BAK1. The sobir1 mutant was generated by CRISPR/Cas9 previously 22 . PsRLK6 ECD -induced ROS production, activation of MAPK and the PTI marker gene CYP71D20 were measured in BAK1-silenced and sobir1 plants. The results showed that the ROS production was remarkably compromised in BAK1-silenced plants and sobir1 mutants compared to GFP-silenced and wild-type plants ( Fig. 2a-d). The relative expressions of CYP71D20 induced by PsRLK6 ECD protein were reduced by about 12-fold in BAK1-silenced plants and 40-fold in sobir1 mutants, respectively (Fig. 2e, f). Similarly, the MAPK activation was also remarkably compromised in BAK1silenced plants and sobir1 mutants (Fig. 2g, h). We next evaluated whether BAK1 and SOBIR1 are also required for PsRLK6 ECD -induced resistance to P. capsici. Compared with leaves treated with TRV2:GFP, BAK1-silenced leaves are more susceptible to P. capsici and PsRLK6 ECDinduced resistance was blocked through monitoring lesion areas and relative pathogen biomass in BAK1-silenced plants ( Supplementary  Fig. 3a-d). Similar results were observed in the sobir1 mutant, which showed increased disease severity and comprised plant resistance induced by PsRLK6 ECD (Supplementary Fig. 3e-g). Collectively, these results showed that BAK1 and SOBIR1 are both required for PsRLK6triggered immunity in N. benthamiana.

PsRLK6 can trigger immune responses in soybean and tomato
We also tested the elicitor activity of PsRLK6 ECD in the cognate host soybean (Glycine max) along with two nonhost plants tomato and Arabidopsis thaliana. The results showed that 1 μM PsRLK6 ECD protein could trigger ROS burst in soybean and tomato, but not in A. thaliana (Fig. 3a-c). Similarly, the marker gene PR1a was up-regulated in soybean and tomato but not in A. thaliana after being pre-treated for 6 h with PsRLK6 ECD compared to GFP protein ( Fig. 3d-f). To further evaluate the contribution of PsRLK6 ECD to disease resistance in soybean, the etiolated soybean hypocotyls were placed into 50 nM PsRLK6 ECD and GFP protein solution respectively. Twelve hours later, soybean hypocotyls were taken out from the protein solution and inoculated with a piece of mycelium of P. sojae. Compared to the GFP control, pretreatment with PsRLK6 ECD resulted in lighter symptoms in plants (Fig. 3g). Similarly, 50 nM GFP and PsRLK6 ECD protein were used to treat tomato and A. thaliana, and PsRLK6 ECD could induce resistance to P. capsici in tomato ( Fig. 3h) but not in A. thaliana (Fig. 3i). These results suggest that PsRLK6 ECD could induce immune responses in multiple plants including the host plant soybean.
A small region (LRR5-6) of PsRLK6 is sufficient to elicit immune responses in plants To identify the critical area of the PsRLK6 for elicitor activity, we firstly predicted the 3D structure of PsRLK6 ECD by AlphaFold 2 23 . Total of five generated models were provided, and the pLDDT coloring was displayed in Fig. 4a. The best predicted structure of PsRLK6 ECD was presented, wherein the ECD forms a single continuous structure in an arc shaped conformation (Fig. 4a). The inner face of the arc forms a concave surface, the majority of which contain an extended parallel β-sheet. The outer face forms a convex side mostly consisting of various secondary structures such as α-helices, loops and turns. According to the predicted structure, PsRLK6 ECD contains an N-terminal signal peptide domain, an LRR capping domain, seven atypical LRR domains, and an LRR C-terminal domain (Fig. 4b). To test whether the elicitor activity of PsRLK6 ECD is heat stable, we treated the PsRLK6 ECD protein by boiling at 100°C for 10 min. The results showed that boiled PsRLK6 ECD protein still could induce ROS accumulation (Fig. 4c) and disease resistance to P. capsici in N. benthamiana (Supplementary Fig. 4). So, (a )   INF1   GFP   1  2  3  4  5  6  7  8  9  1 0  1 1  1 2   13  14  15  16  17  18  19  20  21  22  23 24     we hypothesized an epitope might be recognized by plants to elicit immune responses. To map the PsRLK6 ECD epitope that is responsible for the elicitor activity, we generated truncated mutant variants of PsRLK6 ECD that carried an intact signal peptide and tested their ability to test the ROS accumulation in N. benthamiana by agroinfiltration (Fig. 4b). As shown in Fig. 4b, the region containing residues 237-281 (M7), corresponding to LRR5-6, fully triggered ROS accumulation in N. benthamiana leaves (Fig. 4b). Furthermore, pathogen inoculation assays confirmed that expression of LRR5-6 in N. benthamiana was sufficient to enhance plant resistance to P. capsici (Fig. 4b).
These results indicate that the LRR5-6 of PsRLK6 ECD is the key region for elicitor function. To further verify whether the immunogenic epitope LRR5-6 could trigger plant immunity, the peptide was chemically synthesized. However, we failed to obtain the final peptide after trying three times. Furthermore, we expressed and purified the LRR5-6 region using E. coli to investigate whether it could elicit immune responses in different plants. The results demonstrated that LRR5-6 could induce ROS burst in N. benthamiana (Fig. 4d), and subsequent experiments confirmed its ability to trigger ROS burst in soybean ( Fig. 4e) and tomato (Fig. 4f). In conclusion, these results demonstrate that two LRR motif of PsRLK6 ECD is sufficient to elicit immune response in multiple plants. PsRLK6 ECD induced ROS burst in soybean (a) and tomato (b) but not in A. thaliana (c). Leaf discs from different plants were assayed for ROS production by measuring the relative light units (RLU) with a luminometer upon GFP protein (negative control), flg22 peptide (positive control) or PsRLK6 ECD treatment for the indicated time points. Data are shown as means ± SD (n = 8 biologically independent samples). d-f The relative expression of PR1a in PsRLK6 ECD infiltrated plant leaves mentioned above. GFP was used as the control for PsRLK6 ECD protein treatment. Soybean Actin, tomato Actin and Arabidopsis ACT2 genes were used as references, respectively (n = 3 biologically independent experiments). g PsRLK6 ECD induced disease resistance to P. sojae. Etiolated soybean hypocotyls were soaked with 50 nM PsRLK6 ECD or GFP proteins for 12 h. Lesion lengths were assessed 48 hpi. Relative biomass of P. sojae in the infected etiolated soybean hypocotyls was measured by qRT-PCR and was normalized to that treated with GFP (n = 3 biologically independent experiments). h Induction of disease resistance to P. capsici induced by spray pretreatment of tomato leaves with 50 nM PsRLK6 ECD for 24 h compared to GFP (n = 3 biologically independent experiments). i Spray pretreatment of PsRLK6 ECD in A. thaliana could not induce disease resistance to P. capsici (n = 3 biologically independent experiments). The experiments (a, b, c) were performed three times with similar results. P values were derived by two-tailed Student's t-test. Scale bars, 1 cm. Source data are provided as a Source Data file.

The elicitor function of RLK6 could be conserved in oomycetes
Since LRR-RLKs are widespread in oomycetes and plants, but not in diatoms, fungi, or metazoans 16 , to investigate the phylogenetic distribution of PsRLK6 homologs, we queried the genomes of twenty-six oomycete, three fungal, two bacterial, and two plant species. A total of 53 homologous protein sequences were obtained in different oomycete species, while no homologs were found in others ( Fig. 5a, b, Supplementary Data 1). Interestingly, PsRLK6 homologs were only found in the Peronosporales but not in Saprolegniales (Fig. 5a, b). Most oomycetes contain no more than three PsRLK6 homologs, while Phytophthora fragariae and Phytophthora cactorum have much more homologs than other species (Fig. 5b). Additionally, the Consurf server 24 was used to analyze the conservation of the structure of obtained 53 homologous protein sequences with PsRLK6 ECD . The results showed that they share a high similarity at the protein level ( Supplementary Fig. 5a). The enlarged image of the key region for elicitor function LRR5-6 also showed a high similarity (Supplementary Fig. 5b). To investigate whether the PsRLK6 homologs from diverse Phytophthora and Pythium species could induce immune responses, we cloned the ECDs of six homologous genes from Phytophthora infestans, Phytophthora capsici, Phytophthora parasitica, Phytophthora nicotianae, Phytophthora cactorum, and Pythium oligandrum (Supplementary Fig. 5c). These homologs were transiently expressed in N. benthamiana to test for their ability to induce ROS accumulation. Similar to PsRLK6 ECD , DAB staining assays showed that all six PsRLK6 ECD homologs could induce ROS accumulation in N. benthamiana (Fig. 5c). Western blot analysis confirmed that all the proteins were successfully expressed at the expected size in N. benthamiana (Fig. 5d). Moreover, expression of these six homologs in N. benthamiana all resulted in remarkably reduced P. capsici biomass compared to the GFP control (Fig. 5e). These results indicated that conserved PsRLK6 homologs from diverse oomycetes could be recognized by N. benthamiana to mount plant immunity.

PsRLK6 can trigger immune responses during P. sojae-soybean interaction
MAMPs often play important roles in the growth, development, and virulence of microbes. However, we previously reported that the PsRLK6 knockout mutants have no obvious phenotypes on growth, zoospore development, and virulence 16 . A transcriptome data showed that PsRLK6 was up-regulated during infection stages 25 , suggesting its potential role during interaction. Therefore, we over-expressed PsRLK6-GFP by PEG-mediated transformation 26 . Immunoblotting analysis using the α-GFP antibody confirmed that the protein was expressed in the mycelia of the OT24 and OT36 lines ( Supplementary  Fig. 6a). All transformants showed normal filamentous growth compared with WT ( Supplementary Fig. 6b, c). To assay the virulence of the transformants, zoospores were inoculated onto etiolated seedlings of soybean. The knockout mutant ΔPsRLK6 showed unchanged lesion length as reported previously 16 , while overexpression of PsRLK6-GFP showed significantly reduced virulence on soybean ( Supplementary  Fig. 6d). Determination of the P. sojae DNA biomass confirmed that virulence of OT24 and OT36 transformants was severely attenuated compared to that of WT and ΔPsRLK6 ( Supplementary Fig. 6e, f). These results indicate that overexpression of PsRLK6 could limit P. sojae infection, likely by inducing defense responses.
Furthermore, we conducted a series of experiments to investigate whether PsRLK6 could elicit PTI immune responses during P. sojaesoybean interaction, including ROS burst, up-regulation of PR1a and MAPK activation. At 24 h post-inoculation (hpi), PsRLK6 overexpression line OT24 exhibited a higher level of ROS accumulation compared to both the WT and knockout mutant, which displayed similar levels of ROS staining as the WT after DAB staining (Fig. 6a). Next, the ROS burst in soybean leaves triggered by different strains was quantified, revealing that PsRLK6 knockout mutants produced a slightly reduced amount of ROS compared to the WT, while overexpression lines generated significantly higher levels of ROS (Fig. 6b,  c). Similarly, the fold change of PR1a expression was lower than that induced by the WT, whereas OT24 overexpression line exhibited an opposite trend (Fig. 6d). Further experiments on MAPK activation of different strains revealed that OT24 exhibited greater MAPK activation compared to WT and ΔPsRLK6 exhibited a little less (Fig. 6e), indicating a potential role of PsRLK6 in inducing MAPK activation of soybean. Taken together, these results show that PsRLK6 can trigger defense responses, though the extent to which it does so during infection of plants with wild-type Phytophthora remains to be determined.
PsRLK6 is required for the oospore development of P. sojae In nature, survival and dispersal of spores are essential for the success of many plant pathogens. The sexual reproduction of P. sojae produces thick-walled oospores, which are important inoculum to trigger a new epidemic in the next growing season 27 . Sexual reproduction is particularly relevant for the homothallic P. sojae 28 . To further explore the biological role of PsRLK6 during the sexual cycle of P. sojae, we firstly investigated the number and morphology of oospores produced by PsRLK6 knockout and overexpression transformants. Interestingly, after culturing on LBA (lima bean agar) medium for 14 days, the knockout mutant generated considerably reduced oospores, while overexpression lines produced slightly more oospores than WT (Fig. 7a, b). The oospores produced by ΔPsRLK6 showed more abnormal morphological oospores while overexpression lines showed less (Fig. 7c). We further determined whether PsRLK6 could up-regulate in oospore development stages. The mycelia grown on the LBA medium for 6, 12, and 20 days produced no oospores, immature oospores and mature oospores, respectively. We found that PsRLK6 is up-regulated about 3-fold and 12-fold in the stages of immature and mature oospores respectively (Fig. 7d). Furthermore, we investigated oospore number and morphology produced by different strains during infection. Due to phenotype of two overexpression lines grown on LBA medium was consistent, we selected OT24 for further study. Oospores were generated on infected root tissue with mycelia at 48 hpi (top row) and 96 hpi (bottom row) stained with lactophenol-trypan blue. The results showed that oospores produced in infected soybean hypocotyl tissue by PsRLK6 knockout mutant was less than WT, while PsRLK6 overexpression transformant (OT24) produced more oospores (Fig. 7e, f). Similar to that grown on the LBA medium, the oospores produced by ΔPsRLK6 during infection showed more abnormal morphological oospores, while overexpression line (OT24) showed less (Fig. 7g). We further determined whether PsRLK6 could up-regulate in infection stages when oospores could be produced. In the late stages of infection, the oospores became mature gradually, and the expression levels of PsRLK6 were significantly increased (Fig. 7h). Taken together, these results indicate that PsRLK6 is essential for the oospore development of P. sojae.

Discussion
To overcome pathogen attacks, plants have evolved an excellent system to recognize non-self MAMPs (microbe-associated molecular patterns) through cell-surface receptors, among which LRR-RLKs are the largest family of RLKs in plants 29 . Interestingly, LRR-RLKs are also  , 311-357). The corresponding ROS accumulation (n = 20 biologically independent samples) and disease resistance to P. capsici (n = 3 biologically independent experiments) were shown in the right panel. Bars indicate mean ± SD. The statistical analyses were performed with two-way and different letters show significant difference. c ROS accumulation induced by infiltration of heat-treated PsRLK6 ECD in N. benthamiana leaves. The PsRLK6 ECD protein was treated at 100°C for 10 min. Ratios indicate the proportion of infiltrated sites that developed the ROS accumulation phenotype (n = 8 biologically independent samples). LRR5-6 region of PsRLK6 ECD induced ROS burst in N. benthamiana (d), soybean (e) and tomato (f). Leaf discs from N. benthamiana, soybean and tomato were assayed for ROS production by measuring the relative light units (RLU) with a luminometer upon PBS buffer (negative control) or LRR5-6 treatment for the indicated time points (n = 8 biologically independent samples). The experiment repeated three times with similar results. Data are shown as means ± SEM. Scale bars, 1 cm. Source data are provided as a Source Data file.
widespread in oomycetes and play important roles in development and stress [15][16][17] . Remarkably, here we found a P. sojae LRR-RLK (PsRLK6) and its homologs could be detected by plants to activate patterntriggered immunity.
Sexual reproduction is a key process in the infection cycle of oomycetes, which produce oospores that are important inoculum to trigger a new epidemic in the next growing season 30 . However, the molecular mechanism of sexual reproduction in oomycetes is poorly understood. Only very few genes have been reported to regulate the oospore development of oomycetes. In Pythium ultimum, knockout mutants of PuM90, a Puf family RNA-binding protein, showed significantly defective in oospore formation with empty oogonia or oospores larger in size with thinner oospore walls 31 (e) (d) 16 PuFLP mRNA level to facilitate oospore formation 31 . In P. infestans, silencing of a gene that encodes loricrin-like protein blocked oospore wall formation 32 . As for P. sojae, YPK1 (a serine/threonine protein kinase-encoding gene) 33 , YKT6 (a soluble N-ethylmaleimide-sensitive factor attachment protein receptors) 34 , GK5 (a G-protein-coupled receptor with a phosphatidylinositol phosphate kinase domain) 35 , IMPA1 (a conserved importin α) 36 , and N-glycosylation in HSP70 37 also play important roles in oospore production while the mechanisms of regulating oospore development remain largely elusive. Here, we showed that PsRLK6 plays an indispensable role in oospore production and morphological development. Knockout of PsRLK6 led to reduced oospore number, increased ratio of abnormal oospores. During infecting soybean, ΔPsRLK6 produced much less mature oospores compared to WT. Although knockout of PsRLK6 does not affect the zoospore development and virulence 16 , impaired oospore production and survival rate in soybean residues likely could not guarantee sufficient inoculum to trigger a new epidemic in the next growing season in the field. Microbe-associated molecular patterns (MAMPs) are highly conserved molecules and have an essential function in microbial fitness or survival within the microbiome 3 . These molecules are usually involved in the growth, development, or pathogenicity of microbes. Classical MAMPs are usually structural molecules, such as bacterial flagellin, peptidoglycan, lipopolysaccharides, oomycete glucans, and fungal chitin 3 . Many virulence secretory proteins are also MAMPs, such as NLP 38 , XEG1 39 PcEXLX1 40 . The reproduction of fungi and oomycetes is vital for the infection cycle and virulence variation, but only a few reports indicate that molecules involved in this process were identified as MAMPs. Zoospore of Phytophthora and conidiospore of fungi could directly elicit plant defense during infection 41,42 . Meanwhile, the MAMPs VmE02 43 and VdNLP1 38 regulate the conidiation of fungal pathogens Valsa mali and Verticillium dahliae, respectively. In this study, we first reported a protein that regulates the sexual reproduction of Phytophthora was recognized by plants. And we reveal that the transmembrane protein of pathogens also could be recognized as a MAMP.
The formation of heteromeric PRR complexes is important to activate intracellular signaling for immunity 44 . LRR-RLKs recruit the coreceptor BAK1 upon ligand binding, while LRR-RLPs require another co-receptor SOBIR1 besides BAK1 45 . PsRLK6-triggered immune responses depend on both BAK1 and SOBIR1 in N. benthamiana, suggesting that PsRLK6 is likely recognized by a plant LRR-RLP. In animals, transmembrane LRR proteins regulate synapse formation and function in the vertebrate nervous system by directly interacting with membrane proteins of neighboring cells 46 . During infection, pathogens release cell-wall degrading enzymes to enter the host's apoplast, and host plants also secret enzymes to degrade the cell wall of pathogens to limit infection 47,48 . In pathogen-host interactions, LRR-containing proteins could also mediate the infection cycle. For example, Listeria monocytogenes, a bacterial pathogen that causes gastroenteritis, abortions, and meningitis, produces about 25 different LRR-containing internalin proteins 49  demonstrated that the LRR domains of cell-surface located internalin proteins bind directly to distinct human receptors to promote the internalization of bacteria into mammalian cells 49 . Thus, in the plant apoplast battleground, P. sojae LRR-RLKs might interact with plant receptors to mediate cell-to-cell communications.
Another possible scenario is that the ECD of PsRLK6 is cleaved by the plant and then released into the apoplast. In animals, controlled proteolysis is a well-known general mechanism that regulates the function of transmembrane receptors. Proteolytic cleavage of the extracellular portion of transmembrane proteins at or near the cell surface is referred as ectodomain shedding. It can be either constitutive or stimulus-induced and has been demonstrated for a lot of animal proteins, including cell adhesion molecules, growth factors, and receptors 50,51 . Prominent examples are receptor tyrosine kinases Taken together, we speculate that PsRLK6 ECD might be released into the host's apoplast to be detected by the plant immune receptor. Plant RLKs and RLPs form dynamic complexes in a liganddependent manner, which is essential for signaling activation 54,55 . The Arabidopsis LRR-RLKs show a complicated interaction network revealed by a high-throughput screen 19 . Likewise, we previously showed that P. sojae LRR-RLKs could form various complexes by splitluciferase assay 16 . Indeed, cell surface transmembrane proteins of animals interact directly to mediate cell-to-cell communications 46 . Here, we reveal that a P. sojae LRR-RLK is likely sensed by a plant LRR-RLP that forms a complex with LRR-RLKs BAK1 and SOBIR1 to activate plant immunity, suggesting that the pathogen receptor could also form complexes with plant receptors. The parasitic weed Striga gesnerioides produces an LRR effector to promote host colonization 56 . Therefore, it's interesting to further determine other P. sojae LRR-RLKs that could interact with plant receptors to disturb plant immunity.
The primary way to control plant diseases is using chemical fungicides, however, which threaten the environment and food safety. Microbe-associated molecular patterns (MAMPs) are environmentally friendly biological factors that enhance plant resistance to pathogens 57 . Here, we show that PsRLK6 induces immune responses in soybean and tomato, and the elicitor activity is heat-stable. Thus, PsRLK6 is a promising bio-stimulant for the control of plant diseases. Moreover, immune receptors that recognize MAMPs can be used for plant-resistance breeding. For instance, interfamily transfer of the LRR-RLK EFR that recognizes bacterial EF-Tu proteins in tomato and rice confers broad-spectrum resistance to bacteria 57 . Since RLK6 homologs that have elicitor activity are widespread in oomycete pathogens, further characterization of the corresponding receptor of RLK6 will provide resistance to these pathogens.
In summary, we report that an LRR-RLK from the oomycete pathogen P. sojae is essential for sexual reproduction by regulating oospore development. PsRLK6 triggers BAK1-and SOBIR1-dependent immune responses, representing a novel type of MAMP that shows the cell communication between cell surface receptors from plant and pathogen.

Microbial cultures
The Phytophthora sojae strains (P6497 and transformants), P. capsici, P. parasitica, P. nicotianae, P. cactorum, and Pythium oligandrum were routinely grown on 10% vegetable (V8) juice agar medium at 25°C in the dark. The P. infestans strain T30-4 was routinely cultured on the solid RSA/V8 medium at 18°C in the dark. P. sojae mycelia and zoospores were prepared as previously described 16 . PsRLK6 overexpression transformants were generated using the PEG-mediated protoplast transformation 26 . Overexpression lines were confirmed by immunoblot.

Plant growth conditions and infection assays
In this study, Nicotiana benthamiana plants were grown in soil at 23°C with a 16 h light and 8 h dark photoperiod. Soybean plants ( Hefeng 47) were grown in soil at 23°C with a 16 h light and 8 h dark photoperiod. Tomato seeds were germinated on a Petri dish lined with moist filter paper at 25°C. After 5 days, the germinating sprouting seeds were transferred to soil and grown at 25°C under a 16 h light and 8 h dark photoperiod. Arabidopsis plants were grown at 23°C with a 10 h light and 14 h dark photoperiod.
For the P. capsici infection assay in N. benthamiana, leaves with different treatments were collected. Agar plugs of 5 mm diameter excised from fully grown mycelium of P. capsici were placed on adaxial surface of the leaves and incubated in darkness at 25°C. The inoculated leaves were kept in transparent plastic boxes with high humidity and placed in a climate chamber. Lesion areas were measured at 36 h post-inoculation (hpi).
For the P. capsici infection assay in tomato. Tomato plants were sprayed with GFP and PsRLK6 ECD proteins. After 12 h, leaves were excised. Agar plugs of 5 mm diameter excised from fully grown mycelium of P. capsici were placed on the adaxial surface of the leaves and incubated in darkness at 25°C. After three days post inoculation, disease severity and in planta P. capsici biomass was quantified.
For the P. capsici infection assay in Arabidopsis. Arabidopsis Col-0 plants were sprayed with GFP and PsRLK6 ECD protein. After 12 h, leaves were excised. About 150 zoospores of P. capsici were inoculated onto the center of a detached leaves and incubated in a growth room in dark at 25°C. Inoculated leaves were photographed under UV light and the lesion areas were measured at 24 hpi. Relative quantification of P. capsici biomass in infected Arabidopsis leaves was performed to evaluate disease development.
For the P. sojae infection assay,~200 zoospores of each transformant and the wild-type strain were inoculated onto the hypocotyls of etiolated soybean seedlings (susceptible Hefeng47 cultivar). Disease symptoms were scored at 48 h post-inoculation (hpi). At least three replications were performed for infected seedlings of each transformant. All assays were repeated at least three times.
P. sojae growth, oospore production, and staining assays To determine the growth rate, all tested strains were cultured on 10% V8 agar medium at 25°C in the dark. Colony diameters were measured and photographed after seven days. To quantify oospore production, all tested strains were cultured on LBA agar medium in 7 cm petri plates at 25°C in the dark. The oospores produced by different strains were photographed and counted with at least 30 images per strain. Fig. 7 | PsRLK6 regulates the oospore development of P. sojae. a Morphology of oogonia and oospores generated by WT, PsRLK6-knockout (ΔPsRLK6) and PsRLK6-GFP overexpression transformants (OT-24/36). Oospores were generated on LBA medium for 14 days and observed under a light microscope. The arrows indicate the abnormal oospores. The experiment was repeated three times with similar results. b Statistical analysis of oospore number from 14-day-old cultures on LBA medium (n = 44-48 biologically independent samples). c The proportion of oospores with three different morphologies. d Transcript levels of PsRLK6 measured using qRT-PCR in mycelia, IO (immature oospores) and MO (mature oospores) stages. Data are shown as mean ± SD (n = 3 biologically independent experiments). e Oospores were generated on infected root tissue at 48 hpi (top row) and 96 hpi (bottom row) stained with lactophenol-trypan blue. The arrows indicate the abnormal oospores. f Statistical analysis of oospore numbers from infected root tissue at 48 hpi. Data are shown as mean ± SD (n = 10 biologically independent samples). g The proportion of oospores with three different morphologies during infection. Data are shown as mean ± SD (n = 3 biologically independent experiments). h Transcript levels of PsRLK6 measured using qRT-PCR when mycelia were inoculated in etiolated soybean seedlings for 0, 6,12,24,48,72,84,96, and 168 h. The corresponding oospore morphology was shown above. Data are shown as mean ± SD. The experiment was performed three times with similar results. The statistical analyses were performed with two-tailed Student's t test. Different letters represent significant difference. *P < 0.05; **P < 0.01. In box plots in (b) and (f), the center line, box edges and whiskers indicate the median, lower and upper quartiles and the minimum and maximum, respectively. Scale bars, 20 μm. Source data are provided as a Source Data file.
The different morphology of oospores was photographed, and the content ratio was calculated.
Oospores of the homothallic species P. sojae were collected and separated by following the techniques as described previously 58 . Briefly, a polytron was used to homogenize the mating tissues collected from five dishes. The tissues were washed by 50 ml of sterile deionized water using five intervals of 2 min each at 4°C. Then, oospores and hyphae were concentrated by centrifugation for 10 min at 4000 × g, and washed three times in water by spinning at 650 × g for 5 min. To assess the colonization of soybean tissues by oospores, infected epidermal cells were collected at 48 and 96 hpi and soaked in lactophenol-trypan blue [(10 mL lactic acid, 10 mL glycerol, 10 g phenol, and 10 mg trypan blue (purchased from Beijing Solarbio Science & Technology Co., Ltd.), dissolved in 10 mL distilled water], and then the infected epidermal cells were examined under an inverted microscope.
To examine colonization of soybean tissues by oospores of different strains, infected epidermal cells were collected at 6,12,24,48,72,84,96,168 hpi and then soaked in lactophenol-trypan blue to stain for 2 h. After destaining in chloral hydrate and water, cells were examined under a light microscope. The morphology of oospores was observed and the number was measured. Each strain was tested using at least two different preparations of mycelia and five plants for infection. The lesion areas of plants infected by pathogen are measured by ImageJ v1.52 and the figures are made by GraphPad Prism 9 software.

Bioinformatics
The proteomes of different organisms used in this study were obtained from National Center for Biotechnology Information (NCBI) databases with following accession numbers: Phytophthora sojae (GCA_000149755.2), P. infestans (GCA_000142945.1), P. nicotianae To determine the phylogenetic relationships of selected organisms, BLASTP (v2.5.0) comparisons (E value < 1e-5) of all the protein sequences derived from above organisms were used as input into TribeMCL v14.137 59 using an inflation value of 2.0 for clustering. Protein sequence clusters with only one member from each organism were defined as single-copy core proteins (Fig. 5a). The multigene phylogenetic tree was conducted by MEGA 11 60 following the maximumlikelihood based method with 1,000 bootstrap replicates based on the concatenated sequences of single-copy core proteins. Single copy genes used in the phylogenetic analysis was as listed in Supplementary Data 2. To search for PsRLK6 orthologs in other organisms, PsRLK6 was used as the query to search against proteomes above by reciprocal BLASTP with an E-value cut-off of 1e-5. A phylogenetic tree of PsRLK6 and its homologous proteins was constructed following the neighbor-joining algorithm with 1,000 bootstrap replicates using MEGA 11 software to identify PsRLK6 orthologs (Fig. 5b). The multiple sequence alignment analysis was performed using MUSCLE (v3.8.31) tool with default parameters. To analyze the amino acid conservation, we used the Consurf server (https:// consurf.tau.ac.il/consurf_index.php) with the default parameters except uploading a multiple sequence alignment of obtained 53 homologous protein sequences 24 (Supplementary Figs. 5a and b). The structures were visualized in the PyMOL v2.4.0 program.
Protein structure prediction. The structure of PsRLK6 ECD is predicted by ColabFold 23 v1.5.2: AlphaFold2 using MMseqs2 and the detect templates in pdb70 (Fig. 4a). All the ColabFold output was included with the Supplementary Data 3. The structures were visualized in the PyMOL v2.4.0 program.

Plasmid construction
A mixture of cDNAs from P. sojae collected at different infection stages was used as a template to amplify the coding sequences of P. sojae genes using the Phanta Super-Fidelity DNA Polymerase (P501-d1, Vazyme). Genes were cloned into vectors based on homologous recombination technology using the Vazyme ClonExpress II One Step Cloning Kit (C112, Vazyme). For Agrobacterium-mediated transient expression in N. benthamiana, the coding sequences of P. sojae LRR-RLKs, PsRLK6 ECD homologs and truncated versions of PsRLK6 ECD were cloned into the vector pBIN-3xHA. pBIN:INF1 and pBIN:GFP plasmid constructs served as positive and negative controls, respectively. For expression of the protein in Pichia pastoris, PsRLK6 ECD and PsRLK7 ECD without the signal peptide were cloned into pPICZaA vector. For expression of the protein in E. coli, LRR5-6 region of PsRLK6 ECD were cloned into pCold TF vector. To generate overexpression transformants, PsRLK6 was cloned into the pTOR-GFP vector. The primers and constructs used in this study are listed in Supplementary Table 1.

Transient expression in N. benthamiana and DAB staining
Agrobacterium-mediated transient expression assays were performed to test the ROS-inducing activity. The indicated constructs were transformed into A. tumefaciens strain GV3101. The Agrobacterium strains carrying a plasmid were cultured in the LB medium containing antibiotics at 28°C for 16-18 h. And then cells were collected by centrifugation and resuspended in infiltration buffer (10 mM magnesium chloride (MgCl 2 ), 10 mM MES, 200 μM acetosyringone, pH 5.7) in the dark at 28°C for 2 h. The Agrobacterium cell suspension was infiltrated into N. benthamiana leaves using a needleless syringe at a concentration of 0.5 OD 600 . After N. benthamiana leaves were infiltrated with Agrobacterium for 24 h, the infiltrated leaves were soaked and stained with 3,3-diaminobenzidine (DAB, Sigma-Aldrich) solution for 8 h in the dark and then were destained with ethanol before observation.

VIGS Assays in N. benthamiana
For VIGS assays, pTRV1, pTRV2:BAK1, or pTRV2:GFP plasmid constructs were introduced into A. tumefaciens GV3101. The cultured Agrobacterium cells were harvested and resuspended in infiltration buffer to an 0.5 OD 600 . Agrobacterium strains harboring pTRV2:BAK1 or pTRV2:GFP vector combining with that harboring pTRV1 vector were mixed in a 1:1 ratio. The cocultures were then infiltrated into two primary leaves of N. benthamiana at the four-leaf stage. The effectiveness of the VIGS assay was evaluated using the PDS gene as described 61 . The silencing efficiency of BAK1 was validated using RT-qPCR analysis.

Expression and purification of recombinant proteins
The expression host Pichia pastoris KM71H (Muts) (Invitrogen) was cultured on yeast extract-peptone-dextrose medium. BMGY (buffered glycerol-complex medium) at pH 6.5 was used for yeast growth and BMMY (buffered methanol-complex medium) at pH 6.5 was used for induction (Easy Select Pichia expression kit; Invitrogen). P. pastoris transformants were screened for protein induction in 24-well plates as described 62 . Induction of protein expression was performed according to the manufacturer's instructions. Purification of recombinant GFP, PsRLK6 ECD , and PsRLK7 ECD protein from the culture supernatant was performed by affinity chromatography using Ni-NTA Superflow resin. LRR5-6 region were recombinant expressed using E. coli strain BL21 (DE3) followed by induction with isopropyl β-d-1thiogalactopyranoside (IPTG, 0.5 mM; Sangon Biotech, A600168) at 18°C for 16 h. Then purification of recombinant LRR5-6 TF protein from the culture supernatant was performed by affinity chromatography using Ni-NTA Superflow resin. Subsequently, HRV 3 C Protease (Takara, 7360) was employed to cleave the labeled LRR5-6 protein and produce unlabeled LRR5-6 protein.

Measurement of reactive oxygen species
Reactive oxygen species (ROS) production was monitored with an L-012/peroxidase-based assay on leaf discs collected from N. benthamiana, soybean, tomato, and Arabidopsis plants. The leaf discs were floated overnight on 200 μL of ddH 2 O in a 96-well plate. The ddH 2 O was replaced with a working solution [20 μM L-012 (Waco), 20 μg/mL peroxidase (Sigma-Aldrich), 1 μM flg22 (Sangon), or 1 μM purified protein reaction solution]. After the addition of the working solution, the plate was immediately moved to a GLOMAX96 microplate luminometer (Promega, Madison, WI, USA) for measurement of luminescence.
ROS detection in soybean leaves in response to zoospores of different P. sojae strains was monitored with an L-012 /peroxidasebased assays as previous reported with some minor modifications 63 . Briefly, Leaf discs was collected from 2-week-old soybean plants and then floated overnight on 200 μL of ddH 2 O in a 96-well plate. The ddH 2 O was replaced with a working solution [20 μM L-012 (Waco), 20 μg/mL peroxidase (Sigma-Aldrich), zoospores (10000 zoospores per mL) of WT, PsRLK6-knockout (ΔPsRLK6) or PsRLK6-GFP overexpression transformants (OT-24). After the addition of the working solution, the plate was immediately moved to a GLOMAX96 microplate luminometer (Promega, Madison, WI, USA) for measurement of luminescence.

RNA extraction and quantitative PCR assays
Total RNA of all samples was extracted using a PureLink RNA mini kit. Approximately 900 ng RNA was used for reverse transcription with oligo (dT) primers. Then, the cDNA reaction mixture was diluted three times and 2 μL was used as the template in a 20 μL PCR reaction with SYBR qPCR Master Mix (Vazyme). qPCR was performed using an ABI Prism 7500 Fast Real-Time PCR system as per the manufacturer's instructions. The relative quantitative method (2 −ΔΔCt ) was used to evaluate the quantitative variation.

MAPK assays
Proteins were extracted from 5-week-old N. benthamiana leaves after treatment with purified protein. The leaves were then frozen in liquid nitrogen and stored at −80°C. The frozen leaves were ground in liquid nitrogen and homogenized in protein extraction buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 5 mM EGTA, 2 mM DTT, 10 mM sodium fluoride, 50 mM β-glycerolphosphate, 10% [v/v] glycerol, complete proteinase inhibitor cocktail [Roche, Mannheim, Germany], and Phosstop phosphatase inhibitor cocktail (Roche). Phosphorylation of MAPK proteins was detected by immunoblotting with anti-phospho-p44/42 MAPK antibody (1:5,000, Cell Signaling Technology, Danvers, Massachusetts, USA; #9101) according to the manufacturer's protocol. Western blots were stained with Ponceau S to verify equal loading.
MAPK assays were conducted in soybean leaves in response to zoospores (10,000/mL) produced by different P. sojae strains. Leaf discs was collected from 2-week-old soybean plants and then floated overnight on 200 μL of ddH 2 O in a 96-well plate. The ddH 2 O was substiuted with either fresh ddH 2 O or zoospores (10,000 zoospores per mL) of WT, PsRLK6-knockout (ΔPsRLK6) or PsRLK6-GFP overexpression transformants (OT-24) and incubated for 10 min, 20 min, 30 min. Subsequently, protein extraction and MAPK detection were performed as described above.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.